Affinity sensor for detecting specific molecular binding events and use thereof

ABSTRACT

The invention relates to an affinity sensor for detecting specific molecular binding events, for use in the field of molecular biology, e.g., in medical diagnostics, especially in biosensor technology or in DNA microarreay tests. The aim of the invention is to provide an affinity sensor of this type for rapidity, sensitively, specifically, economically and routinely detecting the presence of molecules, especially bioactive molecules, and to provide special applications for an affinity sensor of this type. To this end, the affinity sensor consists of a support substrate which is provided with at least two electrodes. The electrodes are situated equidistantly from each other and cover an area on both sides, at least this area being provided for receiving immobilized specific binding partners which are capable of coupling complementary corresponding binding partners directly or with other specific binding molecules. The area is established with a minimum width b, in such a way that at least one complementary corresponding binding partner which is provided with an electroconductive particle can be received in the area in such a way as to guarantee the possibility of a tunnel-type contact junction forming between the particle and the electrodes in each case. The affinity sensor is used for biomonitoring.

BACKGROUND OF THE INVENTION

The invention relates to an affinity sensor for detecting specificmolecular binding events, as is particularly used in themolecularbiological field, for example, in medical diagnostics, inbiosensor technology or in DNA-microarray technology, and application ofthe same.

Biosensors are solid phase measuring devices that are comprised of atleast one biological receptor, a transducer and a subsequently connectedelectronic unit.

The receptor utilizes biologically active reagents such as, for example,is antibodies for detecting a specific substance such as, for example,antigens. The transduction of detection events into detectable signalsis performed by the transducer, for example, by electrochemical,optical, piezoelectric, or calorimetric methods. Thereby, the couplingof the detection events to the transducer can be carried out indirectlyor directly. In the first case, the detection events modulate a processwhich is detected by the transducer. In the second case, the detectionevents themselves are recorded by the transducer. The transducer isconnected to an electronic unit, for example, to a microprocessorfollowed by modules for signal detection and evaluation.

There are numerous application possibilities for such biosensorsoperating on the basis of molecular detection. These are, among others,in fields of detection and concentration analysis of biomolecules,kinetic and equilibrium analysis of biochemical reactions, control offermentation processes, evaluation of receptor-cell-interactions,clinical analysis, and cell demotion.

The detection of the presence of bioactive molecules will be performedin the case of nucleic acids, for example, by hybridization withspecific and marked nucleic acid probes. The marking of the probes isachieved by enzymatic inclusion of nucleotides that carry radioisotopessuch as, for example, tritium, sulphur-35 or phosphorus-32,non-radioactive molecules such as, for example, digoxigenin or biotinand non-radioactive fluorescent molecules, respectively, such as, forexample, fluoresceinisothiocyanat or 7-amino-4methylcumarin-3-acetate ormetallic particles such as, for example, gold (Nicholl, D. S. T., 1995:Genetische Methoden, Spektrumis Akademischer Verlag Heidelberg, p.24-27).

In the case of antigens, such as peptides or proteins, the detection ofthe presence of bioactive molecules is achieved by specific and markedantibodies. The marking of the antibodies is performed by coupling ofradioisotopes such as, for example, iodine-125 or tritium, totyrosine-residuals and histidine-residuals, respectively, bynonradioactive enzymes, for example, alkaline phosphatase or peroxidase,whereby the enzymatic activity is measured, for example, by theconversion of a colorless product into a colored one, by nonradioactiveenzymes, for example, haematin which effects the chemiluminescentreaction of hydrogen peroxide and luminol, by nonradioactive enzymes,for example, luciferase which effects bioluminescence by means ofphosphorized luciferin, or by metallic particles such as, for example,gold (Liddell, E. and Weeks, I., 1996: Antikoerpertechniken, SpektrumAkademischer Verlag Heidelberg, p. 87-107).

The signals from the various marker-molecules used will be evaluated byradio-chemical or electrochemical methods, by optical, piezoelectric, orcalorimetric methods for indicating molecular detection events. Thereby,the size of the marker-molecules which emit single signals will lie inthe nanometer area.

The optical and electrochemical methods for representing molecularbinding events are the currently most utilized ones.

The problem of the various optical methods is, that the sensitivity andthe spatial resolution of the signals emitted by the individualmarkermolecules is too low for many applications, that the bindingbetween two links of a specific molecular binding pair cannot bedetected, and that the signals are very often superimposed by anunspecific background. These problems of image generating methods canonly be eliminated in part by an experimental amplification of thesignal or by a computer aided statistical image analyzing method.

The technical limits of the current automation of the image analyzing onthe basis of chip technology lies in a read-out of various microarrayspots. Most of the available technologies are based on detection offluorescence marked binding pairs, which are held in a specific mannerto a surface of a chip, whereby the fluorescence detection is performedby an optical read-out of reactive centers of microarrays. Theapplication of fluorescent or chemiluminescent samples is therebyutilized just as in the conventional method described hereinbefore andis combined with the CCD-imaging (Eggers, M. et al., 1996: ProfessionalProgram Proceedings, in Electro '96. IEEE, New York, N.Y., USA, 364 pp.;Heller, M. J., 1996: IEEE Engineering-in-Medicine-and-Biology-Magazine15: 100-104), whereby also here the mentioned problems of theconventional image analyzing occur and a binding between two links of aspecific molecular binding pair cannot be detected.

The detection of the presence of bioactive molecules can also beobtained by an electrochemical approach by various methods, apart fromthe commonly used optical methods.

The measurement of redox potency variations in biomolecules is awell-known possibility, which is accompanied by specific binding events,for example, on enzymes. Thereby, the redox potential variations aremeasured by way of a single electrode, which is provided with molecules,and a reference electrode (Heller, A., 1992: Electrical connection ofenzyme redox centers to electrodes, J. Phys. Chem. 96: 3579-3587).

The disadvantage of this method lies in the fact that only one singleelectronic event occurs for one biomolecular binding event, whereby thevariation of the redox state, which is effected, lasts only for a shorttime, so that the detection of each individual binding event had to takeplace flash-like. This is not possible. The signal obtained is onlycumulative so that rare binding events cannot be detected by this astechnology.

A further possibility for detecting the presence of bioactive moleculesin an electrical way is to use biosensors in the form of specialmeasuring electrodes. Such special measuring electrodes generally arecomprised of a (strepto)-avidin coated electrode, whereby the(strepto)-avidin has the property to specifically bind biotin molecules.In this way it is possible to detect peptides, oligonucleotides,oligosaccharides and polysaccharides as well as lipides which are markedwith biotin or biotin-derivatives, respectively to couple these asligands to the (strepto)-avid-layer. In the latter case, the biotinmolecules are the coupling elements. Generally, these biosensors allowdetection of antibody/antigen binding pairs, antibody/partial antigenbinding pairs, saccharide/lectin binding pairs, protein/nucleic acidsbinding pairs, and nucleinic acids/nucleinic acids binding pairs. Thedetection of the biochemical events occurring at the special measuringelectrode takes place in a similar way to that of the before describedtechnology based on redox system, namely, by measuring the potentialvariations across a single electrode compared to a reference electrode(Davis, et al., 1995: Element of biosensor construction; Enzyme Microb.Technol. 17: 130-1035).

A substantial disadvantage of this conventional biosensor technology isthe inherent low sensitivity of the measurements at across the measuringelectrodes that cannot be eliminated in that the ligands in aninfinitely great density are bound to the measuring electrode, forexample, by use of a dextran layer. Due to the additional deposition of,as for example, a dextran layer and due to the spatial arrangement ofthe ligands, the concentration of ligands on the electrodes is indeedraised up to the sixfold compared to a ligand single layer, but adetection of rare binding events or even of a binding between twoelements of a special molecular binding pair is not possible.

Further known possibilities are:

-   -   the anchoring of specific antibodies on a semiconductor gate of        a field-effect transistor, whereby a variation in the charge        distribution and, hence, in the circuit of the field-effect        transistor is obtained by the selective binding of antigens to        the special antibody layer;    -   the immobilizing of special antibodies on the surface of an        fiber, whereby measurable optical phenomena such as, for        example, interfering waves and surface plasmons appear due to        the selective binding of antigens to special antibody layers at        the site of intersection between the fiber optics and the        liquid;    -   as well as the method of surface plasmon resonance, in which, at        a definite angle of incidence of light, the refractive index of        a medium is, due to the selective coupling of antigens,        measurably varied at a metal-coated glass body which is provided        with specific antibodies (Liddell E. and Weeks, 1., 1996:        Antikoerpertechniken, Spektrum Akademischer Verlag        Heidelberg, p. 156-158).

The disadvantage of these methods is that rare binding events cannot bedetected by these technologies.

At present there are only a few methods available which allow a rapiddetection of bindings between molecules at low concentrations or evenwith single molecule pairs (Lemieux, Bertrand et al., “Overview of DNAchip technology.” Molecular Breading 4: 277-289, 1988), though thebiochemical process of the binding pair formation with biosensors, forexample, the hybridization of two nucleotide strands or the binding ofantibodies to antigens itself runs very quickly, that is, within thearea of seconds; biochips can be provided with binding molecules, forexample, with specific oligonucleotides (U.S. Pat. No. 5,445,934) orspecific proteins (U.S. Pat. No. 5,077,210) so that a chip technologywill be possible (Osborne, J. C., 1994: Genosensors. Conference Recordof WESCON/94. Idea/Microelectronics. IEEE, New York, N.Y. USA: 434 pp.;Eggers, M. D. et al., 1993: Genosensors, microfabricated devices forautomated DNA sequence analysis. Proc. SPIE-Int. Soc. Opt. Eng. 1998),by which the presence of definite biomolecules can be detected within afew minutes, for example, the presence of genes by use of specificoligonucleotide probes or antigens by use of specific antibodies, and bywhich great prod are indited in the field of biology or medicine,particularly as concerns genetic investigations (Chee, M. et al. 1996:Accessing genetic information with high-density DNA arrays. Science 274:610-614).

A very promising approach as concerns the detection of binding eventsbetween nucleic acid bindings has recently been given by the utilizationof the dielectric relaxation frequencies of the DNA to distinguishbetween hybridized and non-hybridized samples (Beattie et al. 1993. ClinChem. 39: 719-722). The detection of the differences in frequencies,however, requires equipment which still is very expensive and which,moreover, still is far from being utilized as a matter of routine.

Furthermore, there is known another way to electronically distinguishhybridized samples from non-hybridized ones, which consists indetermining the speed of the electron movements along the DNA strands(U.S. Pat. No. 5,780,234). This determination is based on the fact thatthe arrangement of the pi-electron orbits in the double-ended DNA causesthe electrons to move faster in double-stranded DNA, that is, inhybridized DNA than in single-stranded DNA (Lipkin et al., 1995:Identifying DNA by the speed of electrons Science News 147, 117 pp.). Toallow for a determination of these electron movements, the target has tobe positioned exactly between two molecules. One of these molecules hasto be chemically modified in such a way that it acts as an electrondonor and the other one such, that it acts as an electron acceptor, sothat there is a flow of electrons via electrodes measurable.

This expensive method has the disadvantage that it limits itsapplication to the detection of single-sanded nucleic acids fragments ofa defined length and that it is not suited for further biomolecules.

Furthermore, one of the methods for an electrical detection of particlesis known from Bezryadin, A., Dekker, C., and Schmid G., 1997:“Electrostatic trapping single conducting nanoparticles betweennanoelectrodes.” in Applied Physics Leers 71: 1273-1275, in whichnanoparticles are captured in a gap formed by electrode in that avoltage is applied across the electrodes and the capturing of theparticles is detected by way of the flow of the current. In contrast tothe binding events of biomolecule pairs there is no specific biochemicalbinding of the nanoparticles, but the particle is bound to the electrodegap by the electric field.

There is also known from a work by Braun, E., Eichen, Y., Sivan, U., andBen-Yoseph, G., 1998: “DNA templated assembly and electrode attachmentof a conducting silver wire.” in Nature 391: 775-778, that DNA moleculescan be held between two micro structurized electrodes and these moleculeonly exhibited an electric conductivity after having been silver coated,whereby this conductivity has nothing to do with specific biochemicalbinding events of biomolecule pairs.

Alivisatos, A. P., Johnson, K. P., Peng, X., Wilson, T. E., Loweth, C.J., Bruchez Jr. M., P. and Schulz, P., G., 1996: “Organization ofnanocrystal molecules using DNA” in Nature 382: 609-611, generatedcomplexes from short single-stringed DNA-molecules and theircomplementary single-stringed DNA-molecules marked with gold-particlesin solution and deposited these on a TEM-grid with a carbon film for acharacterization by electron microscope. An electric characterization,however, of the molecule pair binding did not take place.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an affinity sensorfor detecting specific molecular binding events, which in a rapid,sensitive, specific way detects the presence of molecules in routineoperation at low expenditures, in particular the presence of bioactivemolecules, as well as to provide for special applications of such anaffinity sensor.

According to the invention, the object is realized by the features ofthe claims. More specifically, an affinity sensor consists of a base onwhich electrodes are disposed in a spaced apart relation capturing aarea that is provided with immobilized specified binding partners,

which specifically couple complimentary associated binding partners,whereby said binding partners carry electrically conductive particles,so that there can be formed an electrically conductive contact betweenthe electrodes and in this way the variation of the electrical

resistance is detectable, when there is a potential applied across theelectrodes, as well as the presence of single or a plurality ofcomplementarily at binding partners, carrying electrically conductiveparticles.

DETAILED DESCRIPTION OF THE INVENTION

The invention will be explained hereinafter in more detail by virtue ofschematical embodiments under reference to the drawings. There is shownin:

FIG. 1 an affinity sensor for detecting specific molecular bindingevents,

FIG. 2 a schematical representation of the affinity sensor for detectingspecific molecular binding events,

FIG. 3 a cross-sectional view of an embodiment of the affinity sensorfor detecting specific molecular binding events,

FIG. 4 a plan view of an embodiment of the affinity sensor in the formof an affinity chip, and

FIG. 5 a sectional view along the plane A—A of the affinity chiprepresented in FIG. 4.

DETAILED DESCRIPTION

An affinity sensor for detecting specific molecular binding events shownin FIGS. 1 and 2, is comprised of a carrier subsonic 1 which is providedwith electrodes 2 enclosing an area 4 ta is provided with immobilizedspecific binding partners 5. Thereby the area 4 represents adiscontinuity in an electric circuit that includes an amplifier circuit8, which can be part of a microchip 9, as well as a measuring andevaluating unit 3, whereby in the present example the electrodes 2,which limit the area 4, are connected to the electric circuit and definea minimum width b of the area 4. The specific binding partners 5 arecape of coupling complementarily associated binding partners 6specifically and directly or via further specific is binding molecules7, whereby the complementarily associated binding partners 6 includingelectrically conductive particles 62 are directly coupled or coupled viabinding molecules. The area 4 is, by the arrangement of the electrodes2, so dimensioned in its width and effective height to detect thecoupling of the immobilized specific binding partners 5 to thecomplementarily associated binding partner 6 which carry theelectrically conductive particles 62 or, via further specific bindingmolecules 7, with the complementarily associated binding partners 6which carry the electrically conductive particles 62. Provided that thespecific binding partners 5 are realized by molecules of a nucleic acidprobe species, the complementarily associated binding partners 6, whichcarry the electrically conductive particles 62, by nucleic acids and theelectrically conductive particles 62 by nanoparticles of a size of 20nm, then the minimum width b of the area 4 is 25 nm and its effectiveheight 20 mm.

The coupling of the specific binding partners 5 in the area 4 to thecomplementarily associated binding partners 6 carrying the electricallyconductive particles 62 effects, when there is applied a voltage acrossthe electrodes 2 (refer to FIG. 1), the motion of the electrons via anelectron transport barrier in such a way that the electricallyconductive particles 62 bridge the area 4 so that electrons tunnel fromparticle 62 to particle 62 and to the electrodes 2, as a result thereofa permanent variation of the electric resistance across the area 4between the electrodes 2 can be measured by aid of the post-connectedamplifier circuit 8 in combination with the measuring and evaluatingunit 3.

The measurements can also be performed in a humid environment, inparticular by aid of a gel layer, instead of measuring in a dry state.

In order to enhance the electric conductivity of the area 4 between theelectrodes 2, which is achieved by way of the complementarily associatedbinding partners 6 in cooperation with the electrically conductiveparticles 62, already known electron-transfer-mediators or effectivediffusing electron donors and electron acceptors can be used, such aswater soluble ferrocene/ferricinium, reducible and oxidizable componentsfrom organic salts, cobaltocenes, hexacyanides and octacyanides ofmolybdenum, tungsten, and iron, respectively, macrocycles and chelatingligands from the transition metals such as cobalt, ruthenium, andnickel, including Co(ethylenediamine)3- and Ru(ethylenediamine)3- andtrisbipyridyland hexamine-complexes from transition metals such as Co,Ru, Fe, and/respectively, organic molecules such as 4-4′-bipyridines and4mercaptopyridines, which are free in solution or present in a geldeposited on the carrier substrate 1 or in a polymer deposited on thecarrier substrate 1. When a known gel-based matrix immobilizationutilizes nucleic acids as specific binding partners 5 then, due to athree-dimensional structure of the polymer, it exhibits an advantagethat a greater number of capturing ligands are immobilized on the smallsurface section of the area 4. By using a highly porous hydro-gel, thehybridization rate, for example, of the nucleic acids which are thespecific binding partners S and the complementarily associated bindingpartners 6, which carry the electrically conductive particles 62, isincreased and lies within areas as they are known for nucleic acids insolution.

The affinity sensor shown in FIGS. 3 and 4, which is in the form of aaffinity chip, is characterized in that the electrodes 2 are designed asmicro-electrodes 21, which are arranged in two p each, capturing arespective affinity area 41. Thus, a matrix of affinity areas 41results, which is adapted to simultaneously and electrically detect inthe different interspaces 4 a plurality of various couplings.

Thereby, the individual affinity areas 41 are designed in aninterdigital electrode structure arranged upon a chip surface 42. Thechip surface 42 consists of silicon or glass upon which, for example, adielectric oxide layer is provided. Due to the digitally branchedmicroelectrodes 21, which, for example, can be manufactured to yield theshape of comb-like electrodes 22, the areas 4 on the affinity area 41can be defined to have a length within a area of 20 μm. Themicroelectrodes 21 are spaced apart and electrically separated from eachother by an inter insulating layer 24, as shown in FIG. 5, which isprovided at the intersections 23 of the micro-electrodes 21. Thereby andprovided that the specific binding partners 5 are realized by themolecules of a nucleic acid probe species, the complementarilyassociated binding partners 6, which carry the electrically conductiveparticles 62, are nucleic acids and the electrically conductiveparticles 62 are nanoparticles of a size of 20 nm, then the areas 4 havean effective height of 100 nm and a width of 200 nm. Consequently, atleast one coupling, which establishes a contact between themicroelectrodes 21, is achieved between the immobilized specific bindingpartners 5 and the complementarily associated binding partners 6 whichcarry the electrically conductive particles 62. In this example, theimmobilized specific binding partners 5 are capturing ligands in theform of nucleic acid probes and the complementarily associated bindingpartners 6, which carry the electrically conductive particles 62, aretarget molecules in the form of nucleic acids. The oligonucleotideprobes immobilized as specific binding partners 5 are bound to asilanized carrier substrate 1 via an amino group, whereby a probedensity in an order of size of 10,000 molecules per U M2 is attained inthis example. The complementarily associated binding partners areoligonucleotides in this example, which are marked with as goldparticles, the hybridization conditions depending on the respectivelyused probes.

Alternatively, the affinity areas 41 can be provided with variousimmobilized specific binding partners 5 in sirs, which are respectivelyseparated from each other.

Affinity areas 41 with immobilized specific binding partners 5 andreference areas 43 with immobilized inactive binding partners 51 areprovided on affinity chips, represented in FIGS. 3 and 4, so that themeasurement of the electric resistance between the micro-electrodes 21is carried out as a reference measurement of the electric resistancebetween an affinity area 41 and a reference area 43, whereby themicro-electrodes 21 can be designed as comb-type electrodes 22. Therebythe immobilized specific binding partners 5 and the immobilized inactivebinding partners 51 can be of a thickness which, when covering theelectrodes 21, permits the tunnel effect, rendering the manufacture ofthe chips technologically more easier.

Since the reference area 43 is free from immobilized specific bindingpartners 5, due to the occupation by inactive binding partners 51, thisspace between the two micro-electrodes 21, insulated from each other,represents an electrical barrier so that there does not take place ameasurable electron transfer between them.

The affinity area 41, which in con thereto carries immobilized specificbinding partners 5, binds via the latter and through the coupling eventthe complementarily associated electrical binding partners 6, whichcarry the electrically conductive particles 62, so that as aresult—hereof, by the conducive particles 62, conduction occurs. Thespace of the affinity areas 41 between the micro-electrodes 21, whichare designed as comb-type electrodes 22, is divided into a plurality ofgaps of nanometer width. The nano-gap formed by the electricallyconductive particles 62 result in that an electron transfer is possiblebetween the two contact faces of the micro-electrodes 21 by virtue ofthe tunnel effect, so that the variation of the resistance can bedetected via the amplifier circuit 8 by means of a measuring andevaluating uni 3, when there is a voltage applied across themicro-electrodes 21. In the present example, the voltage applied lies inan order of size of less than one volt.

Alternatively to the measurement of the potential applied across theaffinity area 41 by an electrode system comprised of referenceelectrode, sample electrode and counter electrode, it is also possibleto employ other methods of an electrical detection such as, for example,potentiometric and voltametric measurements.

Standard chemical linker such as, for example, amino-modified ligands,are used to immobilize the specific binding partners 5 and the inactivebinding partners 51, respectively, such as, for example, antibodies ornucleotide probe so that the chemical linkers are bound to the silanizedchip surface 42 and constitute the affinity areas 41 and the referenceareas 43, respectively.

The marking of the complementarily associated binding partners 6 suchas, for example, protein targets or the target nucleic acid, by means ofelectrically conductive particles 62 is performed according to the knownmethods such as, for example, the final marking with markedoligonucleotides, by utilizing ligases.

In the following, the manufacturing of affinity sensors according to thepresent will be described in more detail. In a preferred embodiment theaffinity sensor is comprised of a plurality of areas 4 (also referred toas detection areas), whereby each of which is captured by at least twoelectrodes 2. These detection areas are provided with specific bindingpartners (capture molecules) 5 such as antibodies, fragments ofantibodies or DNA-, RNA- or PNA-oligonucleotides, to which definiteassociated binding partners (target molecules) 6 bind in a specificmanner. The specific binding partners 5 are defined as marked ornon-marked molecules, which can be selected for being bound to thedesired target molecule in the areas 4 of the affinity sensor. To thisend, not only conventional (bio)molecular binding pairs can be utilizedas capturing molecules and as target molecules, but also specificalchemical binding pairs as known from combinatorial is chemistry, whichcan also be utilized as binding pairs within the frame of the invention.The formation of this described specific binding can be understood as aprimary binding event. It is possible to carry out the detection of thisprimary binding in a one-step procedure or in a multi-step procedure,whereby the specific co-immobilization of the material, which transfersthe electrons, for example, the gold particles 62, is carried out in thelast step each. This co-immobilization can be performed by specifickinds or unspecific kinds of molecular interaction, such as ahybridization of probes marked with gold onto the desired-targetmolecule or by a direct marking of the target molecule with theproperties of an electron transfer in such a way that this marking canbe electronically detected. The mentioned coimmobilization is, inprinciple, separated from the primary binding event, in dependence,however, therefrom and can be performed simultaneously. Thus, theco-immobilization or attachment of material, which transfers electrons,to the designated surface of the affinity sensors can be taken as anindirect result of the primary binding. The detection of thisco-immobilization is obtained by an electronic measurement of thevariation of the electric conductivity across the measuring area, thisvariation of the electric conductivity being an indication of thepresence of target molecules. The primary binding ofelectron-transferring material can be exploited to induce secondarydepositions which are adapted to transport electrons. It lies within thescope of the present invention that the specific binding of targetmolecules can be detected by way of a multi-step process, whichcomprises at least one step by way of which electron-transferringmaterial is deposited, this material effecting a reduction of theelectric resistance across the measuring area. It is possible to useorganic or inorganic substances or compounds for the electron conductiveparticles 62. This conductivity, is used for detecting and marking ofthe desired target molecule, that is, for detecting the presencethereof.

In the following and without limiting the present invention theretothere will be described several possibilities of preparation steps formanufacturing an affinity sensor according to the pro invention.

A. To prepare the required electrodes, a silicon wafer having on oneside an oxide layer of about 1 μm thickness is coated by sputtering witha bonding layer, for example, of 3 nm Ti, to the oxide layer and a goldlayer of a thickness of 50-10 nn. To be able to provide for the electrongap width in the lower nanometer area, a multi-layer masking is utilizedfor the micro-structuring. To this end, a coating with a carbon (30 nm)is performed, followed by a coating with a metal combination (Ti andNiCr, respectively, of a thickness of 10 nm). Subsequently, an electronbean resist (150 nm) is deposited by spinning-on. The exposure isrealized by a mix-technology, in the course of which the large-areaelectrodes 2 are generated by means of a shaped-electron-bean exposuredevice and the minute gaps between the electrodes 2 by means of apoint-beam electron-beam exposure device. The structure is transferredto the metal layer by ionbeam etching (IBE) and to the carbon layer by areactive ion-etching (RIE). The transfer of the structure to thegold-layer and the bonding layer is carried out by way of anIBE-process. Finally, the masking layer is removed in an O2 RE-processat a simultaneous surface activation.

In the following, techniques will be described which are based on asilanization of the surface of the chips. Due to this silanization, thesurfaces are activated for binding amino-modified oligonucleotides. Twodifferent methods for the silanization and subsequent immobilizationwill be explained here. Of course, there are also other possibilitiesfor surface activation and immobilization, apart from the silanization.

B.1. Silanization by Application of 3-aminopropyltrimethoxysilane APTES:

The pre-structured chips with gold electrodes, as described by exampleunder A., are purged in an ultrasonic bath and, in sequence inconcentrated nitric acid, in hydrogen peroxide solution (30%) and water,and subsequently dried for 5 minutes at 80° C. Then the chips will beincubi for 2 min. in a 1% silane solution in 95% acetone/water. Afterhaving been washed for ten times in acetone for 5 minutes each, thechips will be dried at 110° C. Then they will be incubated for 2 h in a0.2%-phenylenediisothiocyanate solution in 10%pyridine/dimethylformamide and washed with methanol and acetone. Chipsactivated in this manner can be stored in a desiccator at 4° C. for alonger time.

Subsequently, the linkage of the amino-modified oligonucleotides isperformed, to this purpose a drop of the oligonucleotide solution (2 mMin 100 mM sodium carbonate/sodium bicarbonate buffer) is deposited uponthe chip. The parallel application of small drops of differentoligonucleotides allows a parallelization, for example, by use of anembodiment of the affinity sensor according to FIG. 4. The deposition ofthe mentioned drops call be performed by means of micro-pipettes,spotters or other available techniques suited for the application ofsmall amounts of samples. Then, the chips are incubated in a moisturechamber at 37° C. for about 1-2 h. After removal of the drops the—chipswill be washed with 1%—ammonia solution for one time, and three-timeswith water. Then drying is carried out at ambient temperature.

B.2. A second possibility of silanization is carried out by applicationof 3-glycidoxypropyltrimethoxysilane (GOPS), to this end, as describedunder B 1., the chips are purged and subsequently are treated in anultrasonic bath, each for 12 min. with hexane, acetone and ethanol. Thenthe chips are dried for 5 minutes at 80° C. The silanization is carriedout with 1 mM GOPS in dry toluol at 80*C for 6-8 h. The chips arethoroughly washed with ethyl acetate and are ready for immediate use.

Subsequently, the linkage of the amino-modified oligonucleotidesperformed. To this purpose a drop of the oligonucleotide solution (550μM in 0.1M KOH) is deposited upon the chip and the chip is incubated inthe moisture chamber at 37° C. for 6 h. Again a parallelization, asreferred to under B.1. can be obtained due to the deposition of aplurality of drops with different oligonucleotides. Then the drops areallowed to dry, and then washing is carried out with water at 50° C.under continuous shaking, followed by drying at ambient temperature.

C. In this part of the specification there will be described thepossibility of marking oligonucleotide probes with colloidal gold. Tostart with, there is required a preparation of the thiolatedoligonucleotide, which is carried out as follows: the 3′-alkylthiolmodified oligonucleotides are solid-phase bound to a dithiolcompound laythe manufacturer to protect its functional group. By separation from thecarrier material the functional group will be released and is then inthe active state. The separation takes place in 50 mM DTT(dithiothreitol) in concentrated ammonium hydroxide at 55° C. for 16 h(original solution: 4-8 mg solid-phase bound oligonucleotide, 450 μlwater, 50 μl 1M DTT, 50 μl cc ammonium hydroxide). After incubation theliquid phase is separated from the solid phase (Controlled Pored Glass,CPG) and desalinated by way of column chromatography. Theoligonucleotides are then washed out in reaction buffers. Theconcentration of the single chromatography fractions is then detected byspectrophotometer.

The reaction solution will be incubated at 55° C. for 16 h at 600revolutions per minute in a thermomixer, and then centrifugated for 2-3min. at an acceleration of about 16,000 m/s². Fractions that areprepared in this manner can be stored for more than 4 weeks at −20° C.The binding of the thiolated oligonucleotides to colloidal gold will bedescribed by example in the following:

There are added to 5 ml gold solution (about 17 nM) 2.5 OD (260 nm)alkylthiololigonucleotides, (final concentration 3.6 nM). Subsequentlyto a pre-incubation for 16 h at ambient Thereafter, again acentrifugation takes place for 25 min. at an acceleration of about16,000 m/s². Fractions that are prepared in this manner can be storedfor more than 4 weeks at −20° C. The binding of the thiolatedoligonucleotides to colloidal gold will be described by example in thefollowing:

-   -   There are added to 5 ml gold solution (about 17 nM) 2.5 OD (260        nm) alkylthiololigonucleotides, (final concentration 3.6 nM).        Subsequently to a pre-incubation for 16 h at ambient        temperature, incubation is carried out after a setting to 0.1 M        NaCl/10 mM sodium phosphate buffer (pH 7.0) for 40 h at ambient        temperature. Thereafter, again a centrifugation takes place for        25 min. at an acceleration of about 16,000 m/s². The resulting        pellet is washed with 5 ml 0.1M NaCl/10 mM sodium phosphate        buffer (pH 7.0), followed by a further centrifugation for 25        min. at an acceleration of 16,000 m/s². The re-dispersion is        carried out in 5 ml 0.3 M NaCl/10 mM sodium phosphate buffer (pH        7.0). 40 μl of the aqueous solution with colloidal gold        particles (diameter of 30 nm in the example) obtained in the        above described manner are placed in the area 4 between the        electrodes 2. After drying, electric measurements, which have        been described herein further up, show a linear current-voltage        characteristic which is indicative of an ohmic behaviour of the        aggregated gold colloids in the area under consideration. A        current of 0.3 μA was measured at a voltage of about 0.3 volt        applied across the electrodes 2.

The affinity sensor as, for example, disclosed in connection with FIGS.3 and 4 and in form of the affinity chips, can find a variety ofapplications as, for example, in the molecular biology and in themedical diagnostics where specific bindings of bioactive molecules totheir corresponding binding partners, for example, DNA, proteins, issaccharides are to be determined.

Based on the electrical detection of specific molecular binding events,the affinity sensor allows to perform a bio-monitoring of, for example,molecules, viruses, bacteria, and cells in the most diverse samples, forexample, in clinical samples, in samples of food and from theenvironment such as, for example, from clarification plants, wherebysuch monitoring is performed in a quick, sensitive and specific way.

1. Affinity sensor for detecting specific binding events in response toa sample medium, comprising: a carrier substrate provided with at leasttwo electrodes and having a predetermined area therebetween, saidelectrodes being equidistantly spaced apart from each other andengagingly bordering said area on opposing sides, at least said areahaving immobilized specific binding partners for affinity bindingcomplementarily associated binding partners wherein the specific bindingpartners are nucleic acids; and said area being accessible to saidcomplementarily associated binding partners provided in the samplemedium and having a minimum width adapted for capture of at least one ofsaid complementarily associated binding partners provided with anelectrically conductive particle within said area by affinity bindingwith said immobilized specific binding partners.
 2. Affinity sensor fordetecting specific binding events as claimed in claim 1, wherein saidwidth is under 800 nm.
 3. Affinity sensor for detecting specific bindingevents as claimed in claim 1, wherein the immobilized specific bindingpartners cover said area with a thickness which permits tunnel effects.4. Affinity sensor for detecting specific binding events as claimed inclaim 1, wherein the electrodes are each two micro-electrodes arrangedin a pair, the electrodes being connected to an amplifier circuit withan associated measuring and evaluating unit so that an electric currentflow across the area can be detected when there is a voltage appliedacross the electrodes.
 5. Affinity sensor for detecting specific bindingevents as claimed in claim 4, wherein the electrodes are part of theamplifier circuit and project from out of the latter.
 6. Affinity sensorfor detecting specific binding events as claimed in claim 5, wherein theamplifier circuit is a component of a microchip.
 7. Affinity sensor fordetecting specific binding events as claimed in claim 1, wherein theelectrodes are comb-like structures opposingly meshed, and saidpredetermined area includes affinity areas positioned between thecomb-like structures.
 8. Affinity sensor for detecting specific bindingevents as claimed in claim 7, wherein the comb-like electrodes and theaffinity areas are arranged on a common chip surface.
 9. Affinity sensorfor detecting specific binding events as claimed in claim 8, wherein thechip surface is silicon.
 10. Affinity sensor for detecting specificbinding events as claimed in claim 8, wherein the chip surface is glass.11. Affinity sensor for detecting specific binding events as claimed inclaim 7, wherein the electrodes are arranged in geometrical symmetry tointerdigital structures and said affinity areas are arranged in amatrix, the electrodes are separated from each other at intersections byan insulating layer arranged between the electrodes.
 12. Affinity sensorfor detecting specific binding events as claimed in claim 7, whereinsaid electrodes are micro-electrodes and the micro-electrodes have alength of 0.1 mm, the width of the area is 0.1 μm and its effectiveheight is 0.02 μm as well as the affinity areas is at a 1:10 ratiorelative to the chip surface.
 13. Affinity sensor for detecting specificbinding events as in claim 7, wherein in addition to the affinity areasat least one reference area has immobolized inactive binding partners.14. Affinity sensor for detecting specific binding events as claimed inclaim 7, wherein a number of specific binding partners per unit area onthe individual affinity areas are different.
 15. Affinity sensor fordetecting specific binding events as claimed in claim 7, wherein theaffinity areas carry different specific binding partners.
 16. Affinitysensor for detecting specific binding events as claimed in claim 1, 13,14 or 15, further comprising a plurality of reference areas havingdifferent inactive binding partners.
 17. Affinity sensor for detectingspecific binding events as claimed in claim 1, wherein the specificbinding partners are suited for entering into chemical coordination. 18.Affinity sensor for detecting specific binding events as claimed inclaim 1, wherein the conductive particles are of sizes in the range of0.1 μm to 5 μm.
 19. Affinity sensor for detecting specific bindingevents as claimed in claim 1, wherein the conductive particles consistof metal-cluster compounds.